Methods of treating diabetes

ABSTRACT

A method of treating a patient with diabetes involving administering to the patient a hybrid molecule which contains a cytotoxin covalently joined to interleukin-2 which is capable of binding to interleukin-2 receptor on a cell that contributes to the disease state of the cell and decreasing the viability of that cell.

BACKGROUND OF THE INVENTION

The field of the invention is diabetes.

Diabetes mellitus is a prevalent and degenerative disease characterizedby insulin deficiency, which prevents normal regulation of blood glucoselevels leading to hyperglycemia and ketoacidosis.

Insulin promotes glucose utilization, protein synthesis, formation andstorage of neutral lipids, and the growth of some cell types. Insulin isproduced by the β cells within the islets of Langerhans of the pancreas.

Some individuals with diabetes are not dependent upon the administrationof exogenous insulin, other individuals are completely dependent uponexogenously administered insulin. Insulin-dependence is related to thedegree of destruction of the β islet cells. Diabetic patients that arenot insulin-dependent can be diagnosed as having diabetes if theyexhibit some of the symptoms of the disease, e.g., hyperglycemia, andhave antibodies to insulin or islet cells, or both. Such patients mayprogress to full-blown insulin-dependent diabetes mellitus if they arenot treated.

Insulin-dependent diabetes mellitus (IDDM) is a T cell dependentautoimmune disease. Activated T cells selectively target insulinproducing beta cells and mediate their destruction. In the most severeform of diabetes, the autoimmune reaction causes complete destruction ofβ cells, resulting in an absolute lack of insulin production in theindividual.

The importance of T cells in human diabetogenic autoimmunity isemphasized by the ability of cyclosporine A to cause remission in newonset IDDM (Stiller et al., 1984, Science 223:1362). However,cyclosporin A-induced remissions have not been proven to be permanent,and the chronically administered high doses of cyclosporin A required tomaintain a remission are associated with nephrotoxicity. Thus,cyclosporin A is an unlikely candidate for general clinical use (Drashet al., 1990, in Pediatric Clinics of North America: Current Issues inPediatric and Adolescent Endocrinology 37:6).

Studies in the non-obese diabetic mouse (NOD) indicate that the diseasein mice is similar to IDDM in humans (Makino et al., 1980, Exp. Anim.29:1). Anti-T cell monoclonal antibodies (anti-Thy 1.2 or anti-CD4)prevent disease in NOD mice (Harada and Makino, 1986, Exp. Anim. 35:539;Shizura et al., 1988, Science 250:659) and anti-CD25 has been shown toprevent insulitis in NOD mice (Kelley et al., 1988, J. Immunol. 140:59).However, the action of anti-CD25 antibody was subsequently shown to beblocked by anti-idiotypic antibodies which had been generated in NODmice (Pankewycz et al., 1988, J. Autoimmunity 1:119). T cell clonesobtained from the islets of prediabetic mice with insulitis precipitatediabetes when transferred into prediabetic NOD mice (Pankewycz et al.,1991, Eur. J. Immunol. 21:873; Haskins et al., 1989, Proc. Natl. Acad.Sci. 86:8000).

SUMMARY OF THE INVENTION

The invention features a method of treating a patient with diabeteswhich involves administering to the patient a hybrid molecule whichcontains a cytotoxin covalently fused to interleukin-2, or areceptor-binding portion thereof. The molecule is capable of binding toa cell which contributes to the disease state of the patient andcontains a high affinity interleukin-2 receptor. The molecule is furthercapable of decreasing the viability of the cell, preferably by killingthe cell.

The cytotoxin molecule is preferably a fragment of a peptide toxin whichis enzymatically active but which does not possess generalizedeukaryotic receptor binding activity.

Preferably, the fragment of peptide toxin can be fragment A ofdiphtheria toxin and enough of fragment B of diphtheria toxin to form apore in a cell membrane.

The hybrid molecule is most preferably DAB₄₈₆ IL-2 or DAB₃₈₉ IL-2.

The term diabetes includes patients that are insulin-dependent, that arenot dependent on exogenously administered insulin but that exhibit someof the symptoms of diabetes, for example, hyperglycemia, and that haveantibodies to either islet cells, insulin, or both. Patients that arenot insulin-dependent eventually become dependent as the diseaseprogresses. Accordingly, the invention features methods for thetreatment of patients with each of the levels of diabetes as definedabove.

The invention also features a method of treating a patient with diabeteswherein there is administered a hybrid cytotoxin IL-2 molecule andcyclosporin A. Cyclosporin A is administered either in conjunction withthe hybrid molecule, or is administered after the diabetic condition ofthe patient has substantially improved as a result of treatment with thehybrid molecule. In this method of treatment, cyclosporin A can beadministered at dosage levels that are effective and yet non-toxiccompared to the levels required to treat a patient that does not receivethe hybrid molecule.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and 1B are graphical representation of the effect of DAB₄₈₆ IL-2and DA(197)B₄₈₆ IL-2 on the acquisition of diabetes. Prediabetic NODmice were irradiated (1,000 rad) and injected i.v. with 2×10⁷mononuclear spleen cells from diabetic NOD mice. a) Mice were injecteddaily with vehicle buffer (n=8), 10 μg of DA(197)B₄₈₆ IL-2 (n=8) or 10μg of DAB₄₈₆ IL-2 (n=9) during 4 weeks after adoptive transfer. b) Micewere injected daily with vehicle buffer (n=8), or 5 μg of DAB₄₈₆ IL-2(n=8) during 4 weeks after adoptive transfer.

FIG. 2 is a graphical representation of the effect of DAB₄₈₆ IL-2 on theacquisition of diabetes. Prediabetic NOD mice were irradiated (1,000rad) and injected i.v. with 2×10⁷ mononuclear spleen cells from adiabetic mouse. Mice were treated with vehicle buffer (TBS), 10, 5 or 1μg/day of DAB₄₈₆ IL-2.

INTERLEUKIN-2 AS A TARGETING AGENT

Interleukin-2 (IL-2) or any IL-2 receptor binding derivative thereof canbe used as a targeting agent for a cytotoxin. The DNA and amino acidsequences of IL-2 are known (Tadatsugu et al., Nature 302:305, 1983),and its structure has been predicted by x-ray crystallography(Brandhuber et al., Science 238:1707, 1987). Analysis of geneticallyengineered variants of IL-2 has provided some information concerningwhich residues are important for IL-2R binding (Collins et al., Proc.Natl. Acad. Sci. USA 85:7709, 1988) and bioactivity (Cohen et al.Science 234:349, 1989; Collins et al., supra). Variants of IL-2 whichare useful in the invention include deletion mutants (Genbauffe et al.,U.S. Ser. No. 388,557, hereby incorporated by reference) which lack oneor more amino acid residues in the region between residue 74 and residue79 (numbering according to Williams et al., Nucl. Acids Res. 16:1045,1988). These mutants effectively target toxins to IL-2R-bearing cells(Genbauffe et al., supra). Generally, IL-2 variants useful for targetinga cytotoxin must efficiently bind IL-2R and be endocytosed. The abilityof various derivatives to bind to the IL-2 receptor can be tested withan IL-2R binding assay described below.

In designing molecules targeted to cells bearing the IL-2 receptor itmust be recognized that the IL-2 receptor, like other receptors, hasseveral forms; and it may be desirable to target cells bearing one formand not another. The human interleukin-2 receptor has a high-, anintermediate-, and a low-affinity form. The high affinity receptor hasan apparent K_(d) of ˜10⁻¹⁰ M and is composed of two subunits, p55 andp75 (also called p70). When expressed on the cell surface, both the p75and p55 subunits are capable of binding IL-2. The p75 subunitcorresponds to the intermediate affinity receptor (K_(d) ˜8.2×10⁻¹⁰ M),and p55 subunit corresponds to the low affinity receptor (K_(d)˜1-3×10⁻⁸ M). The p75 subunit is expressed on the surface of resting Tcells, natural killer cells, monocytes/macrophages, andlymphokine-activated killer (LAK) cell precursors, while the highaffinity receptor is expressed on activated T- and B-cells.

In the method of the invention it may be desirable to target only cellsbearing the high affinity receptor. In these circumstances, usefulmolecules will eliminate or neutralize cells bearing the high affinityIL-2 receptor at a concentration which leaves cells bearing theintermediate or low affinity receptor largely unaffected. When themolecule, like IL-2 itself, has affinity for all three classes of IL-2receptor, selectivity can be accomplished by administering the moleculeat a concentration which does not permit significant binding to cellsbearing lower affinity receptors. A hybrid molecule may have alteredreceptor affinities compared to IL-2. Such hybrid molecules may be moreor less selective for cells bearing the high affinity IL-2 receptor. Forexample, cells bearing the high-affinity receptor are 500-1000 timesmore sensitive to DAB₄₈₆ IL-2, a fusion protein consisting of part ofdiphtheria toxin and part of IL-2, than are cells bearing theintermediate-affinity receptor (Waters et al., Eur. J. Immunol. 20:785,1990).

A cytotoxin can be attached to an IL-2 derivative in a number of ways.Preferably, an IL-2/toxin hybrid is a hybrid protein produced by theexpression of a fused gene. Alternatively, the cytotoxin and the IL-2derivative can be produced separately and later coupled by means of anonpeptide covalent bond. Linkage methods are described below.

Useful cytotoxins are preferably significantly cytotoxic only whenpresent intracellularly and are substantially excluded from any givencell in the absence of a targeting domain. Peptide toxins fulfill bothof these criteria and are readily incorporated into hybrid molecules. Amixed cytotoxin, a cytotoxin composed of all or part of two or moretoxins, can also be used. Several useful toxins are described in moredetail below.

Toxins

The toxin molecules useful in the method of the invention are preferablytoxins, such as peptide toxins, which are significantly cytotoxic onlywhen present intracellularly. Of course, under these circumstances themolecule must be able to enter a cell bearing the targeted receptor.This ability depends on the nature of the molecule and the nature of thecell receptor. For example, cell receptors which naturally allow uptakeof a ligand are likely to provide a means for a molecule which includesa toxin to enter a cell bearing that receptor. The peptide toxin usefulin the methods of the invention is fused to an IL-2R binding domain byproducing a recombinant DNA molecule which encodes a hybrid proteinmolecule. Such an approach ensures consistency of composition.

Many peptide toxins have a generalized eukaryotic receptor bindingdomain; in these instances the toxin must be modified to preventintoxication of non-receptor bearing cells. Any such modifications mustbe made in a manner which preserves the cytotoxic functions of themolecule (see U.S. Department of Health and Human Services, U.S. Ser.No. 401,412). Potentially useful toxins include, but are not limited to:cholera toxin, ricin, 0-Shiga-like toxin (SLT-I, SLT-II, SLT II_(v)), LTtoxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin,Pseudomonas exotoxin, alorin, saporin, modeccin, and gelanin.

Mixed Toxins

The cytotoxic portion of some molecules useful in the invention can beprovided by a mixed toxin molecule. A mixed toxin molecule is a moleculederived from two different polypeptide toxins. Generally, as discussedabove in connection with diphtheria toxin, polypeptide toxins have, inaddition to the domain responsible for generalized eukaryotic cellbinding, an enzymatically active domain and a translocation domain. Thebinding and translocation domains are required for cell recognition andtoxin entry respectively. The enzymatically active domain is the domainresponsible for cytotoxic activity once the molecule is inside a cell.

Naturally-occurring proteins which are known to have a translocationdomain include diphtheria toxin, Pseudomonas exotoxin A, and possiblyother peptide toxins. The translocation domains of diphtheria toxin andPseudomonas exotoxin A are well characterized (see, e.g., Hoch et al.,Proc. Natl. Acad. Sci. USA 82:1692, 1985; Colombatti et al., J. Biol.Chem. 261:3030, 1986; and Deleers et al., FEBS Lett. 160:82, 1983), andthe existence and location of such a domain in other molecules may bedetermined by methods such as those employed by Hwang et al. Cell48:129, 1987); and Gray et al. Proc. Natl. Acad. Sci. USA 81:2645,1984).

One useful IL-2/mixed toxin hybrid molecule is formed by fusing theenzymatically active A subunit of E. coli Shiga-like toxin (Calderwoodet al., Proc. Natl. Acad. Sci. USA 84:4364, 1987) to the translocationdomain (amino acid residues 202 through 460) of diphtheria toxin, and toIL-2. This three-part hybrid molecule, SLT-A/DTB'/IL-2, is useful in themethod of the invention in the same way as DAB₄₈₆ IL-2 described above.The IL-2 portion of the three-part hybrid causes the molecule to attachspecifically to IL-2R-bearing cells, and the diphtheria toxintranslocation portion acts to insert the enzymatically active A subunitof the Shiga-like toxin into the targeted cell. The enzymatically activeportion of Shiga-like toxin, like diphtheria toxin, acts on the proteinsynthesis machinery of the cell to prevent protein synthesis, thuskilling the cell. The difference between these two types of hybridtoxins is the nature of their enzymatic activities: the enzymaticportion of DAB₄₈₆ IL-2 catalyzes the ADP-ribosylation by nicotinamideadenine dinucleotide of Elongation Factor 2, thereby inactivating thisfactor which is necessary for protein synthesis, while the enzymaticportion of SLT-A/DTB'/IL-2 is a ribonuclease capable of cleavingribosomal RNA at a critical site, thereby inactivating the ribosome.SLT-A/DTB'/IL-2 hybrid would therefore be useful as a treatment for thesame indications as DAB₄₈₆ IL-2, and could be substituted or used inconjunction with it if, for example, a patient's activated T-cellsdevelop a resistance to DAB₄₈₆ IL-2.

Linkage of Toxins to Binding Ligands

The binding ligand and the cytotoxin of useful hybrid molecules can belinked in several ways. If the hybrid molecule is produced by expressionof a fused gene, a peptide bond serves as the link between the cytotoxinand the binding ligand. Alternatively, the toxin and the binding ligandcan be produced separately and later coupled by means of a non-peptidecovalent bond.

For example, the covalent linkage may take the form of a disulfide bond.In this case, if the binding ligand is a protein, e.g., IL-2, the DNAencoding IL-2 can be engineered to contain an extra cysteine codon asdescribed in Murphy et al. U.S. Ser. No. 313,599, hereby incorporated byreference. The cysteine must be positioned so as to not interfere withthe IL-2R binding activity of the molecule. For example, the cysteinecodon can be inserted just upstream of the DNA encoding Pro₂ of themature form of IL-2. The toxin molecule must be derivatized with asulfhydryl group reactive with the cysteine the modified IL-2. In thecase of a peptide toxin this can be accomplished by inserting a cysteinecodon into the DNA sequence encoding the toxin. Alternatively, asulfhydryl group, either by itself or as part of a cysteine residue, canbe introduced using solid phase polypeptide techniques. For example, theintroduction of sulfhydryl groups into peptides is described by Hiskey(Peptides 3:137, 1981). Derivatization can also be carried out accordingto the method described for the derivatization of a peptide hormone inBacha et al. U.S. Pat. No. 4,468,382, hereby incorporated by reference.The introduction of sulfhydryl groups into proteins is described inMaasen et al. (Eur. J. Biochem. 134:32, 1983). Once the correctsulfhydryl groups are present, the cytotoxin and IL-2R binding ligandare purified, both sulfur groups are reduced; cytotoxin and ligand aremixed;, (in a ratio of about 1:5 to 1:20) and disulfide bond formationis allowed to proceed to completion (generally 20 to 30 minutes) at roomtemperature. The mixture is then dialyzed against phosphate bufferedsaline to remove unreacted ligand and toxin molecules. Sephadexchromatography or the like is then carried out to separate on the basisof size the desired toxin-ligand conjugates from toxin-toxin andligand-ligand conjugates.

Assays for IL-2 Receptor Binding

The IL-2R binding ability of various molecules can be measured using anIL-2R assay for high affinity (Ju et al., J. Biol. Chem. 262:5723, 1987)or intermediate affinity receptors (Rob et al., Proc. Natl. Acad. Sci.USA 84:2002, 1987).

Assays for Toxicity

Molecules of the invention can be screened for the ability to decreaseviability of cells bearing the targeted receptor by means of assays suchas those described below.

Toxicity towards IL-2R bearing cells can be tested as follows. CulturedHUT 102/6TG (Tsudo et al., Proc. Natl. Acad. Sci. USA 83:9694, 1986) orYT2C2 (Teshigiwari et al., J. Exp. Med. 165:223, 1987) cells aremaintained in RPMI 1640 medium (Gibco, Grand Island, N.Y.) supplementedwith 25 mM HEPES (pH 7.4), 2 mM 1-glutamine, 100 U/ml penicillin, 100μg/ml streptomycin, and 10% fetal calf serum (Hazelton, Lenexa, Kans.).Cells are seeded in 96-well V-bottomed plates (Linbro-Flow Laboratories,McLean, Va.) at a concentration of 1×10⁵ per well in complete medium.Putative toxins are added to varying concentrations (10⁻¹² M to 10⁻⁶ M)and the cultures are incubated for 18 hrs. at 37° C. in a 5% CO₂atmosphere. Following incubation, the plates are centrifuged for 5 min.at 170×g, and the medium removed and replaced with 100 μl leucine-freemedium (MEM, Gibco) containing 8 μCi/ml (³ H-leucine; New EnglandNuclear, Boston, Mass.) After an additional 90 min at 37° C. the platesare centrifuged for 5 min. at 170×g, the medium is removed, and thecells are collected on glass fiber filters using a cell harvester(Skatron, Sterling, Va.). Filters are washed, dried, and countedaccording to standard methods. Cells cultured with medium alone serve asthe control.

Diphtheria Toxin-based Molecules

Diphtheria toxin can be used to produce molecules useful in the methodsof the invention. Diphtheria toxin, whose sequence is known, isdescribed in detail in Murphy U.S. Pat. No. 4,675,382, herebyincorporated by reference. The natural diphtheria toxin moleculesecreted by Corynebacterium diphtheriae consists of several functionaldomains which can be characterized, starting at the amino terminal endof the molecule, as enzymatically-active Fragment A (amino acids Gly₁ -Arg₁₉₃) and Fragment B (amino acids Ser₁₉₄ -Ser₅₃₅), which includes atranslocation domain and a generalized cell binding domain (amino acidresidues 475 through 535).

The process by which diphtheria toxin intoxicates sensitive eukaryoticcells involves at least the following steps: (i) the binding domain ofdiphtheria toxin binds to specific receptors on the surface of asensitive cell; (ii) while bound to its receptor, the toxin molecule isinternalized into an endocytic vesicle; (iii) either prior tointernalization, or within the endocytic vesicle, the toxin moleculeundergoes a proteolytic cleavage between fragments A and B; (iv) as thepH of the endocytic vesicle decreases to below 6, the toxin crosses theendosomal membrane, facilitating the delivery of Fragment A into thecytosol; (v) the catalytic activity of Fragment A (i.e., thenicotinamide adenine dinucleotide-dependent adenosine diphosphate (ADP)ribosylation of the eukaryotic protein synthesis factor termed"Elongation Factor 2") causes the death of the intoxicated cell. It isapparent that a single molecule of Fragment A introduced into thecytosol is sufficient to block down the cell's protein synthesismachinery and kill the cell. The mechanism of cell killing byPseudomonas exotoxin A, and possibly by certain othernaturally-occurring toxins, is very similar.

DAB₄₈₆ IL-2, a fusion protein in which the receptor binding domain ofdiphtheria toxin has been replaced by a portion of human IL-2 (Williamset al., J. Biol. Chem. 35:20673, 1990; see also Williams et al., ProteinEng. 1:493, 1987), is an example of a molecule useful in the method ofthe invention. This molecule selectively kills IL-2R-expressing tumorcells and lymphocytes (Waters et al., Eur. J. Immunol. 20:785, 1990;Kiyokawa et al., Cancer Res. 49:4042, 1989). Because of its ability tokill activated lymphocytes, DAB₄₈₆ IL-2 has been used to control graftrejection (Pankewycz et al., Transplantation 47:318, 1989; Kickman etal., Transplantation 47:327, 1989) and to treat certain autoimmunedisorders (Forte et al., 2nd International Symposium on Immunotoxins,1990).

DAB₄₈₆ IL-2 is a chimeric molecule consisting of Met followed by aminoacid residues 1 through 485 of the mature diphtheria toxin fused toamino acid residues 2 through 133 of IL-2. Thus, DAB₄₈₆ IL-2 includesall of diphtheria toxin fragment A, which encodes the enzymaticallyactive portion of the molecule, and a portion of fragment B. The portionof fragment B present in DAB₄₈₆ IL-2 does not include the generalizedreceptor binding domain but does include the translocation domain whichfacilitates delivery of the enzymatically active portion into thecytosol.

A second molecule useful in the invention is DAB₃₈₉ IL-2, which issimilar to DAB₄₈₆ IL-2 except that it contains 97 amino acids less thanDAB₄₈₆ IL-2.

Preparation of DAB₄₈₆ IL-2 and DAB₃₈₉ IL-2

DAB₄₈₆ IL-2 and DAB₃₈₉ IL-2 are produced and purified as described inU.S. patent application Ser. No. 07/537,430, filed on Jun. 13, 1990, andcorresponding PCT patent application Ser. No. 91/01282, DAB₃₈₉ -IL-2 wasconstructed by removing a 309 bp HpaII-SphI restriction fragment frompDW24 and replacing it with oligonucleotide linker 261/274 to generateplasmid pDW27. This linker restores fragment B sequences from Pro383 toThr387, and allows for in-frame fusion to IL-2 sequences at thisposition. Thus, in DAB₃₈₉ -IL-2 the 97 amino acids between Thr387 andHis485 have been deleted. ##STR1##

IL-2 Toxin Treatment Blocks Diabetogenic Autoimmunity in NOD Mice

High affinity interleukin-2 receptor (IL-2R) is a feature of recentlyactivated T cells and is not detected on resting T cells (Smith, 1988,Science 240:1169; Teshigawara et al., J. Exp. Med. 165:223). An idealtherapeutic for diabetes should rapidly and selectively destroy theactivated, autoaggressive T cells. It has been demonstrated thatdiabetogenic cells express IL-2R in vivo (Pankewycz et al., 1991, Eur.J. Immunol. 21:873). The data described below demonstrates that specificelimination of IL-2R⁺ T-cells aborts the diabetogenic process. Insummary, NOD mice were treated with the IL-2 fusion toxin (DAB₄₈₆ IL-2).DAB₄₈₆ IL-2 selectively binds to the high affinity IL-2-R heterodimer(Waters et al., 1990, Eur. J. Immunol. 20:785). This fusion toxin exertspotent immunosuppression in vivo by preventing delayed typehypersensitivity (Kelley et al., 1988, Proc. Natl. Acad. Sci. USA85:3980) via selective targeting of antigen activated T-cells (Bastos etal., 1990, J. Immunol. 145:3535) and by prolonging engraftment, oftenindefinitely, of allogeneic heart or islet grafts (Kirkman et al., 1989,Transplantation 47:327; Pankewyzc et al., 1989, Transplantation 47:318).The data described below demonstrate that DAB₄₈₆ IL-2 treatment inhibitsdiabetogenic autoimmunity in NOD mice. In addition, diabetic NOD micetreated with DAB₄₈₆ IL-2 do not bear spleen cells that transfer IDDMinto prediabetic NOD mice.

Materials and Methods

As described above, DAB₄₈₆ IL-2 is the product of a fusion gene in whichthe human IL-2 sequence replaces the codons for the receptor-bindingdomain of diphtheria toxin (Williams et al., 1987, Protein Eng. 1:493).DA(197)B₄₈₆ IL-2, is a mutant form of DAB₄₈₆ IL-2 in which a singleamino acid change at position 52 (glycine to glutamic acid) results in aloss of ADP-ribosyltransferase activity. DA(197)B₄₈₆ IL-2 was used as acontrol molecule. These proteins were purified from cellular extracts ofEscherichia coli. They were free of contamination by endotoxins, weresuspended in a vehicle buffer (Tris Buffer Saline), pH 7.9, and werealiquoted at concentrations of 10, 5 and 1 μg/ml. Recombinant human IL-2was purchased from Biogen Inc. (Cambridge, Mass.).

Adoptive Transfer of Diabetes

Two month-old, female NOD mice (Brigham and Women's Hospital; originalbreeding pairs Jackson Laboratory, Bar Harbor Me.) were lethallyirradiated (1,000 rads) and injected intravenously within 4 hours ofirradiation with 1.5 to 2.0×10⁷ mononuclear spleen cells harvested fromspontaneously diabetic NOD mice that had blood glucose levels of >300md/dl. Administration of this number of cells uniformly induced diabetesin 2 month old female NOD mice by 21 days.

Mice were treated subcutaneously with 10 μg/d, 5 μg/d, or 1 μg/d, ofDAB₄₈₆ IL-2 or with 10 μg/d of DA(197)B₄₈₆ IL-2 or with 0.1 ml of thevehicle buffer. To assess the ability of DAB₄₈₆ IL-2 to preventdiabetogenic autoimmunity, some diabetic NOD were treated with 10 μg/dDAB₄₈₆ IL-2 for 1 week. Spleen cells from treated or control mice wereadoptively transferred to lethally irradiated 2 month old prediabeticNOD which received no further treatment.

Results DAB₄₈₆ IL-2 protects NOD mice from adoptively transferreddiabetes

DAB₄₈₆ IL-2 treatment administered subcutaneously prevents diabetes inpre-diabetic NOD mice adoptively transferred with mononuclear spleencells from diabetic NOD mice. Each of 8 NOD mice injected with a vehiclebuffer became diabetic (measured as a sustained blood glucose levelof >200 mg/dl, i.e., three standard deviations above the mean of theblood glucose level measured in prediabetic NOD mice) within 8 weeks ofadoptive transfer. By comparison, only 1/9 mice injected with 10 μg/d ofDAB₄₈₆ IL-2 became diabetic (p<0.001) in this period, while 4/8 of thosemice injected with DA(197)B₄₈₆ IL-2 remained euglycemic (p<0,007) in thesame period (FIG. 1a). Histologic examination of the group of micereceiving DAB₄₈₆ IL-2 sacrificed at 9 weeks, who remained normoglycemicafter receiving diabetogenic T cells, had minimal numbers of mononuclearinfiltrates within islets (1.2±0.6, n=8). By comparison, 5/8 vehiclecontrol treated mice who became diabetic by 4 weeks were already deadand there were few islets remaining in the 3 diabetic sacrificed mice.Unexpectedly, when DAB₄₈₆ IL-2 or DAB₃₈₉ IL-2 was administeredintravenously to prediabetic NOD mice that had received mononuclearspleen cells from diabetic NOD mice, neither compound had any impact onthe diabetic process.

In a second set of experiments designed to determine if DAB₄₈₆ IL-2targets diabetogenic cells, spleen cells transferred from diabetic donorNOD mice injected with DAB₄₈₆ IL-2 were transferred into recipients andwere found to be incapable of inducing diabetes within 8 weeks afteradoptive transfer (0/4 mice). By comparison, spleen cells from untreateddiabetic donor NOD mice induced diabetes in 8/9 animals within this sameperiod (Table 2). Therefore, elimination of IL-2R bearing cells fromdiabetic donor spleen cells in vivo results in at least partialelimination of autoimmune diabetogenic T-cells.

A third set of experiments was designed to determine whether micetreated for 4 weeks with 5 μg/d DAB₄₈₆ IL-2 would remain permanentlyfree of IDDM. Although the rate of development of diabetes was markedlydelayed in DAB₄₈₆ IL-2 treated mice, the 8 treated mice eventuallybecame diabetic between 8 and 16 weeks after adoptive transfer ofdiabetogenic cells. All control mice became diabetic by 5 weeks afteradoptive transfer (FIG. 1b). Of interest, the level of blood glucose inthe DAB₄₈₆ IL-2 treated mice that eventually became diabetic was alwayslower than untreated mice. For example, at the onset of IDDM, untreatedmice had blood glucose levels of 274±8 mg/dl, while treated mice hadvalues of 231±10 mg/dl (p<0.01). Blood glucose levels continued toincrease in untreated mice and the majority of this group achievedvalues above 300 mg/dl, while mice receiving either 10 or 5 μg/d DAB₄₈₆IL-2 never exhibited values as high as 300 mg/dl. Thus, even though micetreated with 4 weeks of DAB₄₈₆ IL-2 eventually did become diabetic,treatment with this fusion-toxin dampened the severity and rate ofprogression of disease.

Mice treated with IL-2 alone had a similar incidence of diabetes (7/8)as compared to the vehicle control group (5/5), and mice injected withDA(197)B₄₈₆ IL-2 (5/8) also became diabetic (Table 1). Thus, IL-2 aloneor DA(197)B₄₈₆ IL-2 could not account for the beneficial action ofDAB₄₈₆ IL-2.

                  TABLE 1                                                         ______________________________________                                        IL-2 DOES NOT ALTER THE TEMPO                                                 OF DIABETES IN NOD MICE                                                       TREATMENT      INCIDENCE OF DIABETES* (%)                                     ______________________________________                                        IL-2 (2.5 μg/d)                                                                           88                                                             n = 8                                                                         DA(197)B.sub.486 IL-2(10 μg/d)                                                            63                                                             n = 8                                                                         VEHICLE BUFFER 100                                                            N = 5                                                                         ______________________________________                                         *Blood glucose determination at 3 weeks after adoptive transfer of            prediabetic NOD mice irradiated (1000 rad) and injected i.v. with 2           × 10.sup.7 spleen cells from diabetic NOD mice. Subcutaneous            treatments were given daily in a volume of 0.1 ml.                       

                  TABLE 2                                                         ______________________________________                                        PRETREATMENT OF DIABETIC NOD MICE                                             WITH DAB486-IL-2 PROTECTS                                                     FROM ADOPTIVE TRANSFER OF DIABETES                                            PRETREATMENT    ONSET OF DIABETES                                             OF DONORS       (weeks after adoptive transfer)                               ______________________________________                                        none+           3, 3, 3, 4, 4, 5, 5, 8                                        n = 9                                                                         DAB.sub.486 IL-2 (10 μg/d)*                                                                8, 14, >16, >16 (p<0.01)                                      n = 4                                                                         ______________________________________                                         +Young NOD mice were irradiated (1,000 rad) and reconstituted with 2          × 10.sup.7 mononuclear spleen cells from recently diabetic untreate     NOD mice and received no further treatment.                                   *Young NOD mice were irradiated (1,000 rad) and reconstituted with 2          × 10.sup.7 mononuclear spleen cells from recently diabetic NOD mice     that were treated with DAB.sub.486 IL2 (10 μg/day) for one week and        received no further treatment.                                           

Therapy

Generally, the molecules of the invention can be administered byintravenous infusion/injection. They may also be administeredsubcutaneously or intramuscularly. Dosages of molecules useful in themethods of the invention will vary, depending on factors such as the ageof the patient, the severity of diabetes in the patient and the route ofadministration. Patients may be treated with 25 to 300 kU/kg of eitherDAB₄₈₆ IL-2 or DAB₃₈₉ IL-2. Either compound can be administered to apatient daily, or intermittently, or daily for a period of time,followed by intermittent administration.

More than 100 patients have received DAB₄₈₆ IL-2 in Phase I/II clinicalprotocols. The molecule is well tolerated with the maximum tolerateddose (MTD) established by transient asymptomatic hepatic transaminaseelevations in about 30% of patients treated at the MTD. Anti-tumoreffects have been seen in approximately 40% of patients; responses werenoted in B-cell leukemias and lymphomas, cutaneous T-cell lymphoma andHodgkin's disease (LeMaistre et al., Blood 360a:abstract 1429, 1990;Woodworth et al., Fourth International Conference on HumanRetrovirology, 1991). Serum concentrations. of 10⁻⁸ M DAB₄₈₆ IL-2 havebeen safely achieved in patients with IL-2 receptor expressingmalignancies. Significant anti-tumor effects have been observed inhighly refractory leukemia/lymphoma patients and these effects haveoccurred despite the presence of elevated soluble IL-2R levels in allpatients. This observation is consistent with data which suggest thatsoluble IL-2R does not interfere with binding of IL-2 to the highaffinity interleukin-2 receptor. Animal and human studies havedemonstrated that DAB₄₈₆ IL-2 has no general immunosuppressive effect(LeMaistre et al., supra; Woodworth et al., supra).

Experiments indicate that binding and internalization of DAB₄₈₆ IL-2 bycells bearing the high affinity IL-2 receptor occurs within 30 minutesof exposure, resulting in maximal inhibition of protein synthesis withinseveral hours. Therefore, the molecule should be effective even if theserum half-life is rather short.

Clinical Studies in Patients with IDDM

A phase I/II study of safety, tolerability, pharmacokinetics andbiological response of DAB₄₈₆ IL-2 in humans with recent onset IDDM isunderway.

This pilot study was designed to evaluate the safety and tolerability ofDAB₄₈₆ -2 in IDDM patients and to assess pharmacokinetics and immunefunction effects, together with changes in diabetic status as determinedby insulin requirement, C-peptide levels, and control of hyperglycemia.Based on experience in similar studies, such effects can bepreliminarily assessed over a 4 to 6 week period following agentadministration, and thus, onset autoimmune diabetes mellitus provides aclinical model for the evaluation of potential immunologic andanti-diabetic effects of a new therapeutic like DAB₄₈₆ IL-2.

DAB₄₈₆ IL-2 has been administered to individuals over 15 years of agewith symptoms ≦4 months in duration, HLA DR3 or 4 and/or anti-islet cellantibody formation. Patients received a 60 minute intravenous infusiondaily for 7 days in a cohort dose-escalation protocol, evaluating doselevels of 0.025, 0.05, and 0,075 mg/kg. This pilot study has evaluated24 patients, each receiving a single course.

To date, 18 patients are evaluable. The agent has been well tolerated inthis group of patients; there has been mild transient hepatictransaminase elevations in 15 to 20% of patients, two transient episodesof edema and two incidences of mild rash suggestive ofhypersensitivity-like effects. Surprisingly, 8 of these patients (3 inthe 0.025 mg/kg, 3 in the 0.05 mg/kg, and 2 in the 0.075 mg/kg dosegroups) have had a substantial decrease in insulin requirement, togetherwith a sustained increase in C-peptide (≧0.6 nanomolar), and anormalization of glycosylated hemoglobin. Data analysis for the other 6patients is underway.

Further, in these responding patients, the addition of cyclosporin A ata non-nephrotoxic dose (4-5 mg/kg/d) resulted in sustained or evengreater improvement in the response parameters. Since this low dose ofcyclosporin A would not be expected to induce such an improvement (Bach,1989 in Thompson, ed. Cyclosporin A: Mode of Action and ClinicalApplications. London Kleuver Academic Publishers, 181), this observationsuggests that treatment of diabetic patients with an IL-2 fusion toxinsuch as DAB₄₈₆ IL-2 followed by, or in conjunction with, low dosecyclosporin A is a highly effective way to safely administer cyclosporinA to patients with diabetes.

The data described above demonstrate that DAB₄₈₆ IL-2 is capable ofsuppressing IDDM in NOD mice, and that this suppression can beattributed to the ability of DAB₄₈₆ IL-2 to target IL-2 expressing Tcells and kill them. The data described above also demonstrate thatDAB₄₈₆ IL-2 is surprisingly useful in reducing insulin dependence inhuman patients that already have IDDM.

What is claimed is:
 1. A method of treating a patient with diabetes, said method comprising administering to said patient a hybrid molecule comprising a first portion and a second portion joined together covalently, said first portion comprising a cytotoxin capable of decreasing cell viability, said second portion comprising all or an interleukin-2 receptor binding portion of interleukin-2 capable of binding to a high affinity interleukin-2 receptor on a cell, said hybrid molecule being administered to said patient in the presence or absence of cyclosporin A administered at a non-nephrotoxic dosage of 4-5 mg/kg/day until said patient's diabetic condition has substantially improved, following which cyclosporin A, but not said hybrid molecule is administered to said patient, said cyclosporin A being administered at a non-nephrotixic dosage of 4-5 mg/kg/day.
 2. The method of claim 1, wherein said hybrid molecule kills cells bearing said receptor.
 3. The method of claim 1, wherein said cytotoxin is a fragment of a peptide toxin which is enzymatically active but which does not possess generalized eukaryotic receptor binding activity.
 4. The method of claim 1, wherein said cytotoxin comprises fragment A of diphtheria toxin and enough of fragment B of diphtheria toxin to form a pore in a cell membrane.
 5. The method of claim 1, wherein said hybrid molecule is DAB₄₈₆ IL-2.
 6. The method of claim 5, wherein said DAB₄₈₆ IL-2 is administered at a dosage of 25 to 300 kU/kg.
 7. The method of claim 1, wherein said hybrid molecule, is DAB₃₈₉ IL-2.
 8. The method of claim 7, wherein said DAB₃₈₉ IL-2 is administered at a dosage of 25 to 300 kU/kg.
 9. The method of claim 1, wherein said patient is not dependent upon the administration of exogenous insulin.
 10. The method of claim 1, wherein said patient has antibodies to insulin.
 11. The method of claim 1, wherein said patient has antibodies to islet cells.
 12. The method of claim 1, wherein said patient is dependent upon the administration of exogenous insulin.
 13. The method of claim 1, wherein administration of said hybrid molecule is stopped prior to administration of said cyclosporin A.
 14. The method of claim 1 wherein administration of said hybrid molecule occurs in the absence of cyclosporin A administration. 